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Electron Cryomicroscopy
Electron Cryomicroscopy (Cryo-EM) is a means of imaging neural serial sections, usually cut by a cryoultramicrotome, now at an experimental maximum (uniform) sample width-reduction of 40-60 nanometers (nm). Sometimes called cryo-EM or Cryo-electron microscopy it is a form of electron microscopy (EM) where the sample is studied at cryogenic temperatures (generally liquid nitrogen temperatures). CryoEM is developing popularity in structural biology. A version of electron cryomicroscopy is cryo-electron tomography (CET) where a 3D reconstruction of a sample is created from tilted 2D images, again at cryogenic temperatures (either liquid nitrogen or helium). Biological specimens Thin film The biological material is spread on an electron microscopy grid and is preserved in a frozen-hydrated state by rapid freezing, usually in liquid ethane near liquid nitrogen temperature. By maintaining specimens at liquid nitrogen temperature or colder, they can be introduced into the high-vacuum of the electron microscope column. Most biological specimens are extremely radiation sensitive, so they must be imaged with low-dose techniques (usefully, the low temperature of cryo-electron microscopy provides an additional protective factor against radiation damage). Consequently, the images are extremely noisy. For some biological systems it is possible to average images to increase the signal to noise ratio and retrieve high-resolution information about the specimen. This approach requires that the things being averaged are identical (e.g. ribosome particles). Analysis of ordered arrays of protein, such as 2-D crystals of membrane proteins or helical arrays of proteins, also allows a kind of averaging which can provide high-resolution information about the specimen. This technique is called electron crystallography. Vitreous sections The thin film method is limited to thin specimens (typically < 500 nm) because the electrons cannot cross thicker samples without multiple scattering events. Thicker specimens can be vitrified by plunge freezing in ethane (up to tens of μm in thickness) or more commonly by pressure freezing (up to hundreds of μm). They can then be cut in thin sections (40 to 200 nm thick) with a diamond knife in a cryoultramicrotome at temperatures lower than -135 °C (devitrification temperature). The sections are collected on an electron microscope grid and are imaged in the same manner as specimen vitrified in thin film. This technique is called cryo-electron microscopy of vitreous sections (CEMOVIS) or cryo-electron microscopy of frozen-hydrated sections. Tissue is often prepared in vitreous ice, preventing ice crystallization by freezing that would otherwise compromise the hereby possible near-ideal (~99%) structure-integrity. Reference Alcor.org here: http://www.Alcor.org. Electrons are then projected at high velocity and density in a cryogenically (liquid nitrogen or helium) cooled vacuum chamber stabilized from exterior vibration through the flux of an invisible magnetic lense against the fixed target material. The pattern of electron diffusion and defraction read on a sensor-film the opposite side is interpreted as readings used to calculate preceise anatomical characteristics, which become by supercomputer manifest as a detailed digital image. A major drawback to this technique is the threat of radiation exposure - and thus biochemical alteration - of the sample's given electron exposure intensity per square unit, time requisite for bioinformatics-retention (radiation exposure time), and the inverse proportionality of high depth-resolution to the material's superficial radition exposure/damage. Also, this is obviously an invasive procedure, meaning the specimen is of a consciousness legally, though not techically dead; for clarification on the definition of death refer to information on cryonics. Finally, neither serial sectioning (cutting the material - in this case, the brain - into small slices of equal width) nor scanning by electron microscopy are fully automated, often requiring at least partial manual assistance that is tedious and time-consuming to the extreme, making large-scale perception and evaluation impractical given limited funding and general resources. The only exception of this is the nematode, which has the smallest brain known and has been mapped by this method completely. Application of the attained information is of limited practical significance relative to the shortcomings of any stand-alone mechanism (as well as its resultant metadata complex therefore), and is often optimally beneficial when synthesized in hybrid with the many other imaging techniques, borrowing their appropriate specialized data-perception abilities as compensation for its own short-comings. Most commonly, these complimentary imaging operations include x-ray crystallography (x-ray), magnetic resonance imaging (MRI), scanning electron microscopy, and high-power/magnification light microscopy aided by one or multiple of many available and experimental staining techniques, for this purpose primarily in bath and not by injection (so as to avoid inexorable damage to the neuron so imposed upon, or literally into). As the first of these four primary choices of examination are non-invasive they are more commonly employed, especially in psychiatric evaluation, which lacks the need present in computational neuroscience (e.g. for purposes of virtual neural circuitry as a biological prototype for artificial intelligence (A.I.) development) to image the microscopic mechanisms of, and not simply infer the macro-scale electrical and magnetic properties of the brain. However, electron transmission microscopy is the tool of choice for neuroscientists interested in high-resolution imaging of neural networks and the intracellular biochemical processes that comprise the cornerstone of their morphology, ergo the capability of learning characteristic of intelligence.